1. Field of the Invention
The present invention relates to microcapsules sensitized with antibody and an improved method for cell-mediated immunity measurement of lymphocyte using such microcapsules. More particularly, the present invention relates to microcapsules sensitized with antibody to antigen of lymphocyte, and, a method for measuring lymphocyte using the foregoing microcapsules based on cell-mediated immunity and sheep red cells, a method for measuring lymphocyte using at least two kinds of the foregoing microcapsules, based on cell-mediated immunity, which are distinguishable from each other, and further a method for the measurement of B cells of lymphocyte using microcapsules sensitized with antibody to antigen of B cells of lymphocyte.
2. Background of the Invention
The term "cell-mediated immunity" is well established in the art and refers to specific immunity which is mediated by small lymphocytes (cells of 6 to 8 .mu.m in diameter with a deeply-stained nucleus and narrow rim of cytoplasm), and is dependent on the presence of the thymus at birth. Cell-mediated immunity is responsible for reactions such as delayed hypersensitivty, tuberculin test reactions, immunodeficiency, etc. and is important in defense against viral infections and against some bacteria.
To immunologically identify or measure T cells (thymus derived cells, also called lymphoid cells that have been demonstrated to be derived from the thymus) and B cells (bone marrow derived cells; lymphocytes found in the peripheral lymphoid organs, i.e., spleen and lymph nodes, etc. which are known to be derived from the bone marrow, but has not been processed by the thymus) of human lymphocytes in the clinical field, the identification or measurement of T lymphocytes (T cells) has been hitherto performed generally by the E rosette technique; B lymphocytes (B cells) have been identified or measured by detecting Fc receptor (receptor for Fc fragment or crystallizable fragment obtained by papain hydrolysis of immunoglobulin molecules) or C3 receptor (receptor for the third component of complement) according to the EA (short for ovalbumin obtained from the avian egg) or EAC (short for erythrocyte antibody complement) rosette technique or by detecting immunoglobulins at the surface of cell membrane according to the fluorescent antibody technique. These methods are based on most fundamental reactions in cell-mediated immunity; in recent years, these methods have been widely utilized in routine tests as determining a proportion of T cells to B cells that are inevitable for the presumptive observation of the immune function of the patient. However, as long as the prior art methods are utilized, T and B cells must be identified or determined separately using independent plates. In addition, the identification of B cells requires complicated procedures. That is, sheep red cells bind to T cells as they are; accordingly, they should be inactivated to T cells and then chemically changed so as to have a binding site for B cells. These procedures also involve poor detection sensitivity. Furthermore, when the fluorescent antibody technique is utilized, relatively large amounts of a testing sample are required so that physical load of the patient becomes serious, and, not only special equipments or facilities are necessary but also technical skill is required for operations of these methods. Such restrictions are problems encountered in these methods.
Further in the E rosette technique and the EA rosette technique or the EAC rosette technique, red cells of animals are employed and hence, these techniques involve problems that extremely complicated operations are required for the preparation of a red cell suspension and moreover, it is impossible to store red cells for long periods of time.
It has been found that T cells and B cells of lymphocytes and other cells can be identified or measured with a single operation simultaneously in a simple operation using an extremely small quantity of a testing sample as compared to the prior art methods, by performing an immune response using one kind or plural kinds of microcapsules sensitized with antibody to antigen of lymphocytes, alternatively, together with sheep red cells or by bringing such microcapsules into contact with lymphocyte adhered to a microplate, etc., and the present invention has thus been accomplished.
Microcapsules having bound thereto an antigen or antibody at the surface thereof are disclosed in U.S. Pat. No. 4,342,739. However, these microcapsules are utilized for the determination of serum components based on cell agglutination, not for the determination of lymphocytes through the rosette formation techniques.